: This is an application to continue studies on the cellular/molecular mechanisms underlying neuronal growth responses to extracellular signals, the development and maintenance of distinct plasma membrane domains in neurons, and the retrograde influence of postsynaptic muscle cells on the differentiation and functioning of presynaptic neurons during the early phase of synaptogenesis. The studies are focused on the spatial and temporal properties of cytosolic signaling mechanisms and the regulation of membrane trafficking in developing neurons. Cultured neurons and myotubes from Xenopus and neurons from rat hippocampi will be used. The specific aims are to understand 1) the role of Ca2+ and cAMP as second messengers in regulating the rate and direction of neurite extension, 2) early cellular events leading to the turning of growth cones induced by diffusible chemotropic substances, 3) mechanisms by which new membrane material is incorporated into the plasmalemma, 4) mechanisms underlying the formation and maintenance of distinct axonal vs. somatodendritic plasma membrane domains in hippocampal pyramidal neurons, 5) influence of muscle contact on the motility and morphology of growth cones, the localization of plasma membrane proteins, and the trafficking of cytoplasmic organelles, and 6) long-range signaling within the neuronal cytoplasm triggered by local synaptic contacts and trans-synaptic retrograde signals, and consequences on transmitter secretion from other parts of the neuron. Using a combination of electrophysiological, optical, and molecular techniques, including patch-clamp electrophysiology, Ca2+ imaging, photoactivation of caged compounds, and expression of chimeric green fluorescent proteins, the proposed studies will yield novel information concerning the mechanisms responsible for regulating the growth and synaptogenesis of developing neurons.